Method of culturing freeze-dried microorganisms and resultant preparation

ABSTRACT

A preparation of freeze-dried microorganisms and a method for preparing a freezed-dried suspension of microorganisms and culture medium, in which the freeze-dried microorganisms can be directly cultured via addition of setrile, distilled water. The freeze-dried preparation contains a sufficient amount of a colloidal component, such as gelatin, to form the microorganisms into a microbial plug.

This application is a continuation-in-part of application Ser. No.548,418 filed 11/3/83, now U.S. Pat. No. 4,672,037.

BACKGROUND OF THE INVENTION

1. Field of the Invention

This invention broadly relates to a new method of culturing freeze-driedmicroorganisms, as well as to a new method of freeze-dryingmicroorganisms and the product resulting therefrom. The presentinvention particularly pertains to a method of freeze-dryingmicroorganisms in a vessel with nutrients so as to produce afreeze-dried preparation containing viable microorganisms that undergosubstantial logarithmic or exponential growth directly in the vesselupon rehydration with sterile, distilled water. This invention hasutility in diagnostic applications, such as in research and in clinicallaboratories, and as an educational tool.

2. Description of the Prior Art

Freeze-drying has long been considered a simple and economical way topreserve cultures for future experimentation, reference or comparison.Freeze-drying produces a dry preparation generally capable of beingstored for long periods of time while retaining biological activity. Theprocess is broadly described in American Type Culture CollectionMethods, I. Laboratory Manual on Presentation: Freezing andFreeze-Drying, Hatt, H. (ed.), A.T.C.C. (1980) with specific referenceto processing suitable for different microorganisms. Theabove-referenced manual describes both the conventional proceduressuitable for freeze-drying a wide variety of microorganisms andequipment suitable for carrying out the procedures.

When the moisture content of a culture is removed during thefreeze-drying process, molecules are virtually locked in position sothat little or no opportunity exists for alteration of the physical orchemical properties of the product. Obviously, in preparing suchproducts it is of prime importance that the viability of the culture bemaintained. In order to protect the organisms, the freeze-drying processgenerally is carried out in the presence of a variety of cryoprotectantagents, designed to minimize cellular damage and increase survivabilityof microorganisms during the freeze-drying process. Cryoprotectantagents used in the prior art have included glucose,, sucrose, lactose,mannitol, glycerol, dextran, fructose, and other materials such asmonosodium glutamate polyvinylpyrrolidone (PVP), sweet whey solids,dried skim milk, dried whole milk and bovine serum albumin.

It is also common to include a small amount of fresh nutrient media(e.g., nutrient broth) in the concentrated microorganism culture priorto freeze-drying. The nutrient media is used as a biologicallycompatible suspending agent. The nutrient media may also function as acryoprotectant and also provides an immediate food source for themicroorganisms upon their revitalization. The prior art, however, dosenot disclose a process for freeze-drying microorganisms in a vesselwherein the proportion of microorganisms to nutrient media in thesuspension to be freeze-dried is adjusted so that when the freeze-driedpreparation is properly rehydrated with sterile, distilled water, anactive, growing culture is produced in situ.

The prior art always has used a concentration of organisms in relationto the quantity of nutrients added with the suspending liquid such thatstandard rehydration of the freeze-dried preparation with sterile,distilled water produces a non-dormant culture in a substantiallystationary growth phase of development. Rehydration generally isaccomplished by mixing the freeze-dried preparation with suitablenutrient broth, sterile distilled water, etc. Thereafter, propagation ofthe culture normally is accomplished by inoculating freshly preparedgrowth media with all or part of the rehydrated preparation. In otherwords, freeze-dried preparations of microorganisms always have been usedto propagate numerous fresh cultures using freshly prepared, externalnutrient medium rather than being used directly in the vessel in whichthey are stored to propagate a single culture in situ.

It is an object of the present invention to provide a simple method ofestablishing a growing culture in a vessel, such as in a small vial,from a freeze-dried preparation of microorganisms.

It is also an object of the present invention to provide a method ofreconstituting a freeze-dried preparation of microorganisms whicheliminate the need for preparing or supplying fresh nutrient medium withthe freeze-dried preparation in order to develop an actively growing andmultiplying culture from the freeze-dried preparatin of microorganisms.

It is another object of this invention to provide a method offreeze-drying microorganisms and nutrients in a vessel so that anactive, growing culture is produced in situ merely by rehydrating thefreeze-dried preparation with sterile, distilled water.

It is still another object of this invention to provide a vessel havinga specific freeze-dried composition of microorganisms, nutrients andoptionally a cryoprotective agent, such that simple addition of a properamount of sterile, distilled water to the vessel produces an active,growing culture therein.

These and other objects which will become apparent from the followingdescription are provided by the present invention.

SUMMARY OF THE INVENTION

In a first aspect, the present invention comprises a method ofestablishing a growing culture in a vessel comprising (a) providing asealed vessel containing a freeze-dried preparation of microorganismsand nutrients, the number of viable microorganisms and the amount ofnutrients in said freeze-dried preparation being such that the nutrientsare sufficient to support substantial logarithmic growth of the viablemicroorganisms, and (b) unsealing said vessel and adding water theretoto initiate the logarithmic growth of said viable microorganisms.

In another aspect, the present invention comprises a sealed vesselcontaining a freeze-dried preparation of microorganisms and nutrients,the number of viable microorganisms and the amount of nutrients in saidfreeze-dried preparation being such that upon the addition of water tosaid vessel the nutrients are sufficient to support substantiallogarithmic growth of the viable microorganisms in said vessel.

In yet another aspect, the present invention comprises a method offreeze-drying microorganisms and nutrients in a vessel to produce afreeze-dried preparation therein comprising:

a. growing a culture of the microorganisms in a nutrient medium;

b. forming a cell concentrate from said culture;

c. adding fresh nutrient medium to the cell concentrate to form a cellsuspension;

d. freeze-drying the cell suspension in a vessel to produce afreeze-dried preparation in said vessel, and

e. providing an amount of nutrients in said fresh nutrient medium sothat said nutrients are sufficient to support substantial logarithmicgrowth of the viable microorganisms in said freeze-dried preparation.

In a further aspect, the present invention provides a method ofestablishing a growing culture in a vessel comprising:

a. growing a culture of the microorganisms in a nutrient medium;

b. forming a cell concentrate from said culture;

c. adding fresh nutrient medium to the cell concentrate to form a cellsuspension;

d. freeze-drying the cell suspension in a vessel to produce afreeze-dried preparation in said vessel;

e. providing an amount of nutrients in said fresh nutrient medium sothat said nutrients are sufficient to support substantial logarithmicgrowth of the viable microorganisms in said freeze-dried preparation;and

f. rehydrating said freeze-dried suspension in said vessel with water sothat the viable microorganisms in said freeze-dried suspension, uponproper incubation, undergo substantial logarithmic growth.

DETAILED DESCRIPTION

The present invention relates to a method of freeze-dryingmicroorganisms and nutrients in a vessel so as to produce a freeze-driedpreparation that undergoes substantial logarithmic growth directly inthe vessel upon rehydration with water. In order to avoid adding anyother organism or potentially toxic material to the freeze-driedpreparation, the preparation preferably is rehydrated using sterile,distilled water. Accordingly, the use of sterile, distilled water willbe emphasized throughout the remaining specification.

As used herein, the phrase "substantial logarithmic growth" means thatthe ending cell population (i.e., the cell population at the stationaryphase) is at least about 10 times greater than the initial rehydratedviable cell population. Preferably, the amount of nutrients and numberof viable microorganisms in the freeze-dried preparation are adjusted sothat a population increase of at least 100 times occurs in therehydrated suspension. The phrase "a concentration of viablemicroorganisms substantially below the concentration that would prevailduring the microorganisms stationary growth phase" is a relatedexpression which means that the initial cell population in therehydrated suspension is at least 10 times less than the ending cellpopulation (i.e., the cell population at the stationary phase).

The term "culture" describes a population of microorganisms cultivatedor grown in a nutrient medium.

Also as used herein the phrases "fresh nutrient medium," "suitablenutrient medium" and the like mean an aqueous solution or suspension ofnutrients which are necessary and sufficient to support the growth ofthe microorganism being freeze-dried.

Freeze-drying or lyophilization is generally recognized as an effectiveprocedure for preserving microorganisms. In this process, microorganismsare dried while they are frozen. In Carrying out the method of thisinvention, the microorganisms initially are grown in any suitablenutrient medium, preferably using the best nutrient medium for theparticular genus and species of microorganism to be freeze-dried.Suitable nutrient or growth medium generally contains, in addition to acarbon source, the usual and necessary nutrients, such as, for example,a nitrogen source; sources of sulfur and phosphorus; inorganic materialssuch as trace metals; growth factors; oxygen and carbon dioxide. Thenutrient medium generally is prepared from commercially availablematerials suitable for the microorganism being freeze-dried, e.g., BactoNutrient Broth (Difco Co., Detroit, Mich.).

The composition of a suitable aqueous nutrient medium or basal mediumsuitable for cultivating a particular microorganism strain will readilybe recognized by one skilled in the art. Moreover, it is specificallycontemplated that the concentration of specific nutrients in thenutrient medium can be selected so as to influence the growth of themicroorganisms in any desired manner. The medium can be prepared, forexample, by dissolving or suspending the proper ingredients (nutrients)in distilled water, followed, if necessary, by sterilization and aseptictransfer into the culture. As noted above, generally a commerciallyavailable, pre-prepared nutrient source is used. The microorganisms areincubated, preferably within their optimum temperature range, oftenuntil they reach the maximum stationary phase. In the practice of thisinvention, incubation times vary from one microorganism strain toanother, depending on the growth medium, temperature and growthcharacteristics of the microorganism. For example, vegetative materialgenerally is harvested in the mid- to late-logarithmic phase of growth.

The method of this invention is applicable to a wide variety ofmicroorganisms, including algae, fungi (including yeast), bacteria andprotozoa. As noted above, suitable nutrient media and growth conditionsapplicable to particular microorganism strains are evident to oneskilled in this art. For example, bacteria generally are capable ofgrowing over a wide range of physical conditions and are capable ofutilizing many different nutrients, but as is well-known, optimal growthrequires certain specific conditions for a given species. Suitablecommercially available nutrient media for several organisms areidentified in the examples included hereafter.

After harvesting the incubated culture, a cell concentrate is preparedfrom the culture of microorganisms. The concentrate can be obtained, forexample, by centrifuging the previously grown culture, and removing theaqueous supernatant. This step removes any metabolic by-products whichmay interfere with the microorganism's survival during the freeze-dryingprocess and/or materials which may inhibit growth after subsequentrehydration. Other methods of preparing a cell concentrate will beapparent to one skilled in the art. Afterwards, the cell concentrate issuspended in fresh, sterile nutrient medium.

The cell concentrate is diluted with a predetermined volume of a knownconcentrate of fresh nutrient medium to produce a cell suspensionGenerally, the nutrient medium employed for preparing the cellsuspension has the same nutrient components as the medium initially usedto grow the culture, but the nutrients are present at a higherconcentration. Previous comments concerning the nutrient medium contentused to grow the microorganism are equally applicable with respect tothe fresh nutrient medium used to form the cell suspension.

The present invention has as an objective the formation of afreeze-dried preparation of microorganisms and nutrient medium in avessel, such that simple addition of a proper amount of sterile,distilled water thereto produces an actively growing culture in situ. Inorder to determine the volume and concentration of nutrient mediumneeded in any given case it initially is necessary to fix (ascertain)the concentration of cells in the cell concentrate and determine whatproportion or number of those cells will survive the freeze-dryingprocess, i.e., the proportion of viable microorganisms in thefreeze-dried preparation. These values can readily be determined byroutine experimentation, e.g., by using direct or indirect cell countingtechniques, and generally remain quite constant for a given strain ofmicroorganism which is cultivated, harvested (i.e., preparation of cellconcentrate) and freeze-dried using closely reproduced procedures. Avolume and concentration of the fresh nutrient medium then is selectedso that the resulting freeze-dried preparation produced from the cellsuspension contains a number of viable microorganisms, i.e., a number ofmicroorganisms which survive the freeze-drying process, in mixture withan amount (content) of nutrients such that when the freeze-driedpreparation is rehydrated with a suitable volume of sterile, distilledwater the rehydrated suspension contains a concentration of viablemicroorganisms substantially below the concentration that would prevailduring the microorganisms stationary growth phase in admixture with aconcentration of nutrients sufficient to support substantial logarithmicgrowth of the microorganisms.

The rehydrated culture must contain a suitable concentration ofnutrients to support substantial logarithmic growth of themicro-organisms after the freeze-dried preparation has been rehydratedwith sterile water. Generally, the rehydrated suspension will contain aconcentration of viable microorganisms less than or about 10⁷ cells perml. The general relationship between the cell concentrate, the volumeand concentration of the fresh nutrient medium used to prepare the cellsuspension, and the suitable volume of sterile, distilled water used torehydrate the freeze-dried preparation is represented in the followingequations and best illustrated by the following hypothetical example.Examples of specific microorganisms are presented in detail here after.

    V.sub.fn [k f.sub.1 C.sub.c V.sub.c ]/[C.sub.sd f.sub.2 ]  (1) and

    VH.sub.2 =[kf.sub.1 C.sub.c V.sub.c ]/C.sub.sp             ( 2)

    f2=[V.sub.H20 /V.sub.fn ]=[C.sub.fn /C.sub.rs ]            (3)

where

V_(fn) =the predetermined volume of fresh nutrient medium

V_(H2O) =a suitable volume of sterile, distilled water used forrehydrating the freeze-dried suspension

f₁ =the fraction of cells in the cell concentrate which will survive thefreeze-drying process, i.e., the fraction of viable cells in thefreeze-dried preparation

C_(c) =the concentration of microorganisms in the cell concentrate

V_(c) =the volume of the cell concentrate

C_(sp) =the stationary phase concentration of microorganisms in therehydrated suspension

f₂ =the ratio of the concentration of nutrients in the fresh nutrientmedium (C_(fn)) to the concentration of nutrients desired in rehydratedsuspension (C_(rs))

k=a constant, equal to or greater than 10, which represents themagnitude of growth desired in the rehydrated suspension

Micoorganism strain A when incubated in its optimum nutrient mediumreaches a stationary phase population of about 10⁷ cells per ml. A cellconcentrate prepared from this culture by a standard centrifugationprogram has a volume of 0.1 ml (V_(c)) and contains about 5×10⁷ cellsper ml (C_(c)). When this concentrate is freeze-dried using a standardfreeze-drying procedure, about 80% of the cells will survive a givenperiod of storage under appropriate conditions (f₁ =0.8). As a result,under these particular conditions the cell concentrate contains about4×10⁷ viable cells per ml, i.e. cells which will survive thefreeze-drying process.

In order for this microorganism to undergo a substantial logarithmicgrowth when rehydrated it is necessary that the rehydrated suspensioncontain less than 10⁶ cells per ml, (i.e., an increase in organismpopulation of at least 10 times; k=10), provided that the incubationconditions for the rehydrated suspension (including the concentrate ofnutrient mddium) closely parallel the conditions initially used to growthe culture (recall that the assumed stationary phase population isabout 10⁷ cells per ml). In-other words, in this case C_(sp) =10⁷. Toobtain a rehydrated suspension containing less than 10⁶ cells per ml,the freeze-dried suspension must be diluted with about 4 ml of sterile,distilled water (Equation (2)), thereby fixing a suitable volume ofrehydration water for the freeze-dried preparation. Obviously a littlemore or less rehydration water is possible and to some extent willeffect population dynamics. However if too much rehydration water isused then the concenration of nutrients will be too low to supportgrowth. On the other hand, if too little rehydration water is added thenthe resulting concentration of nutrients will be toxic to themicroorganisms. Based on this disclosure one skilled in this art willreadily recognize the permissible range in the amount of rehydrationwater added to initiate substantial logarithmic growth of themicroorganisms.

Prefereably, the concentration of nutrients in the fresh nutrient mediumadded to the, cell concentrate is greater than would normally be used togrow the organism in the rehydrated suspension so as to minimize thequantity of water that must be removed during the freeze-drying process,i.e., f₂ ≧1 . For example, nutrients at a level of at least two timesthe standard is preferred (f₂ =2). The quantity (volume) of freshnutrient medium required can then be calculated from Equation (1).

As discussed above, the fresh nurient medium preferably is prepared suchthat the optimum composition for the particular genus and species beingfreeze-dried is produced upon rehydration. In this hypothetical example,with f₂ =2, the cell concentrate would be diluted with about 2 ml ofdouble strength fresh nutrient medium to produce the cell suspension.

It should be noted, the preceding equations need not be applied rigidly;values calculated therefrom are preferably used only as guidelines.These equations are included herein primarily to illustrate and explainthe present invention more completely. For convenience, a nephelometeror spectrophotometer can be used to ensure preparation of a cellsuspension with a proper (approximate) cell concentration.

In the conventinal freeze-drying procedure for preservingmicroorganisms, much higher concentrations of microorganisms are used inthe cell concentrate relative to the quantity of nutrients added withany suspending liquid, so that after considering the number of cellswhich survive the freeze-drying process, a conventionally rehydratedsuspension contains a concentration of microorganisms at or near thestationary phase.

Normally, the cell suspension of the present invention will also containa protective menstrum or cryoprotectant. The cryoprotectant increasesthe survivability of the micro-organisms through the freeze-dryingprocess. In the broad practice of this invention, any of the widevariety of cryoprotectants disclosed in the prior art can suitably beemployed, and one skilled in this technology will readily recognize thatcertain cryoprotectants are more effective with particular strains ofmicroorganisms. Certain of the cryo-protectants, such as dried skimmilk, may produce a clouded culture upon rehydration. Suchcryoprotectants are not preferred when it is desired to monitor thegrowth of the rehydrated suspensions using optical techniques such asspectrophotometry, nephelometry, or turbidimetry. In such instances, acryoprotectant such as sucrose, or bovine serum albumin which addslittle to the optical character of the rehydrated culture, is generallypreferred.

After preparing the cell suspension, generally also containing acryoprotectant, the suspension is freeze-dried in a suitable vessel.Preferably, the cell suspension is freeze-dried as soon as possibleafter its preparation. Generally, a wide variety of glass and in somecases plastic ampules and vials typically are used. Transparent vesselsare particularly preferred as these permit visual inspection and opticalanalysis of the growing, rehydrated suspension. The vessel typically hasa volume below about one liter, and vessels with a volume below 100 mlgenerally are used. The vessel is cleaned, sterilized and properlylabelled before being used.

A complete discussion of procedures used to freeze-dry the cellsuspension is beyond the scope of the present invention and proceduresreadily available to the prior art have been found suitable. Oneprocedure is described in detail in the examples that follow. Generally,the above-prepared suspension will be freeze-dried by initially freezingthe culture at about -40° C. for about one hour (e.g., in a commercialfreeze-drier) followed by evacuating the sample to low absolutepressure. During evacuation, which occurs over an extended time period,the temperature of the preparation is allowed to rise slowly to ambientconditions. Any standard freeze-drying equipment can suitably be used inthe practice of the present invention. For a detailed description ofproper procedures and equipment suitable for freeze-dryingmicroorganisms in the broad practice of the present invention, pleaserefer to the ATCC Laboratory Manual on Preservation: Freezing andFreeze-Drying referenced earlier, the disclosure of which isincorporated in its entirety herein.

After the last desired amount of moisture has been removed, the vesselcontaining the freeze-dried preparation of microorganism and nutrientsis properly sealed and is now ready for storage. The vessel contains aquantity of freeze-dried microorganisms and freeze-dried nutrients suchthat upon proper rehydration and incubation an active microbial cultureis produced in situ which culture undergoes substantial logarithmicgrowth from an initial cell population to an ending cell population.

The vessel containing the freeze-dried preparation can be stored underambient conditions, although storage at a constant temperature belowabout 5° C. will prolong the length of survival of the freeze-driedmicroorganisms. One skilled in this art will recognize whether aparticular microorganism strain requires additional special storageconditions, otherwise standard storage practices are proper.

In accordance with the method of this invention, a growing culture ofthe freeze-dried microorganisms can now be re-established simply byrehydrating the freze-dried preparation with a suitable volume ofsterile distilled water directly in the storage vessel. The seal of thevessel is broken and a quantity of water necessary to adjust theconcentration of nutrients in the rehydrated suspension to aconcentration suitable for supporting substantial logarithmic growth ofthe viable microorganisms in the freeze-dried suspension is added to thevessel. In this way, the proper concentration of nutrient medium isestablished in situ. The vessel containing the rehydrated suspension isthen incubated under conditions, e.g., light, oxygen, temperature, etc.,appropriate for the species of microorganism involved. After a shortlag, the cell population enters the exponential or logarithmic phase ofrapid growth. During this phase of growth, the cells divide steadily ata substantially constant rate. The cell population undergoes substantiallogarithmic growth from an initial cell population to an ending cellpopulation, corresponding to the constant cell population existing atthe stationary phase of growth. The trend toward cessation of growth isgenerally caused by the depletion of some nutrients. In some cases itmay be caused by the production of toxic by-products of growth.Generally, after reaching the stationary phase the cell population willremain constant for a certain period of time.

By freeze-drying the microorganisms culture (cell suspension) in atransparent vessel, and by employing a cryoprotectant that does notadversely affect the optical characteristics of the rehydratedsuspension, the progress of the cell population can readily be monitoredby optical techniques such as spectrophotometry, nephelometry, andturbidimetry. In this way, the product of this invention is useful, forexample, as an educational tool for teaching students about cellpopulation dynamics, reproduction and growth. As an educational aid, theproduct of this invention is supplied as a kit. The kit comprises apackage which includes one transparent vessel containing thefreeze-dried preparation of microorganisms and nutrients and a secondvessel containing a suitable amount of sterile, distilled water forrehydrating the freeze-dried preparation. The product of this inventionalso has application in research and in chemical laboratories, forexample, in diagnostic applications, or in any application where anactively growing culture generally is prepared from a freeze-driedpreparation.

As a check on diagnostic procedures, clinical laboratories generallyrequire a known culture of the specific bacteria being examined ordetected in order to verify the accuracy of their procedures and thesuitability of their reagents. Consequently, there is a demand for areadily available and economic source of viable, standard-reactivemicroorganisms. The present invention not only adequately satisfies thisneed but does so in a simple, convenient and economical manner.

The present invention eliminates the need for preparing or supplyingfresh sterile nutrient medium each time an actively growing culture isprepared from a freeze-dried preparation of microorganisms.Consequently, both the time and expense of preparing an active culturefrom a freeze-dried sample is substantially reduced.

The following examples are included for illustrative purposes only andare not itended to limit the scope of this invention.

EXAMPLE 1

A pure culture of Bacillus subtilis a specific strain available from theAmerican Type Culture Collection number 6051 was grown as a test tubeculture in Bacto Nutrient Broth DIFCO 0711. When the cell populationreached late log, the cells were harvested and a cell concentrate wasprepared by centrifuging the culture. The cells then were resuspended indouble strength Bacto Nutrient Broth DIFCO 0711 containing 12% (v/v)sucrose and 5% (v/v) bovine serum albumin to obtain a cell suspension ofabout 10⁷ cells per ml. The cell concentration was verified using aspectrophotometer.

Thereafter, 1.0 ml of the cell suspension was added to a 4.0 mltransparent glass vial. The vessel was plugged with a butyl rubberstopper and the culture was freeze-dried by placing the vial on theprecooled (-40° C.) shelf of a Virtis 25 SRC-MS sublimator and allowingthe product to cool to -40° C. (product temperature monitored). Thesublimator chamber was evacuated to less than 50 microns (Hg), condenserat -55° C.; shelf warmed to -30° C. and sublimation allowed to proceedfor about 68 hours. Shelf temperature was then raised to 30° C. anddrying continued until product temperature reached 20° C. Vials wereback filled with sterile N₂ and stoppered. Part of the freeze-driedsamples were then stored at about 37° C. for 80 days as part of anaccelerated storage test. Storage for 80 days at 37° C. is equivalent tostorage at 25° C. for 1200-1600 days.

Thereafter, the freeze-dried preparation was removed from storage, theseal on the vessel was broken and 3 ml of sterile water was added to thevessel to rehydrate the freeze-dried preparation. Upon properincubation, the rehydrated culture exhibited substantial logarithmicgrowth from an initial cell population of 2.4×104 cells/ml.

EXAMPLE 2

A pure culture of Escherichia coli, a specific strain available from theAmerican Type Culture Collection number 11775 was grown as a test tubeculture in Nutrient Broth DIFCO 0711. When the cell population reachedlate log, the cells were harvested by centrifuging the culture and thenresuspending the cells in double strength Bacto Nutrient Broth DIFCO0711 containing 12% (v/v) sucrose and 5% (v/v) bovine serum albumin toobtain a cell concentration of about 10⁷ cells/ml. The cellconcentration was verified using a spectrophotometer.

Thereafter, 1.0 ml of the cell concentrate was added to a 4.0 mltransparent glass vial. The vessel was plugged with a butyl rubberstopper and the culture was freeze-dried using the same procedure asExample 1.

After the same accelerated storage period as in Example 1 thefreeze-dried preparation was removed from storage, the seal on thevessel was broken and 3.0 ml of sterile water was added to the vessel torehydrate the culture. Upon proper incubation, the rehydrated cultureexhibited substantial logarithmic growth from an initial cell populationof 2.9×105 cells/ml.

EXAMPLE 3

A pure culture of Serratia marcesens, a specific strain available fromthe American Type Culture Collection number 8195 was grown as a testtube culture in Bacto Nutrient Broth DIFCO 0711. When the cellpopulation reached late log, the cells were harvested by centrifugingthe culture and then resuspending the cells in double strength BactoNutrient Broth DIFCO 0711 containing 12% (v/v) sucrose and 5% (v/v)bovine serum albumin to obtain a cell concentrate of about 10⁷ cells/ml.The cell concentration was verified using a spectrophotometer.

Thereafter, 1.0 ml of the cell concentrate was added to a 4 0 mltransparent glass vial. The vessel was plugged with a butyl rubberstopper and the culture was then freeze-dried using the same procedureas Example 1.

After the same accelerated storage period as in Example 1, thefreeze-dried preparation was removed from storage, the seal on thevessel was broken and 3.0 ml of sterile water was added to the vessel torehydrate the culture. Upon proper incubation, the rehydrated cultureexhibited substantial logarithmic growth from an initial cell populationof 6.3×103 cells/ml.

EXAMPLE 4

A pure culture of Rhodototorula rubra, a specific strain available fromthe American Type Culture Collection number 9449 was grown as a testtube culture in Bacto YM Broth DIFCO 0003-01-6. When the cell populationreached late log, the cells were harvested by centrifuging the cultureand then resuspending the cells in double strength Bacto YM Broth DIFCO0003-01-6 containing 12% (v/v) sucrose and 5% (v/v) bovine serum albuminto obtain a cell concentrate of 1.7×10⁷ cells/ml.

Thereafter, 1.0 ml of the cell concentrate was added to a 4.0 mltransparent glass vial. The vessel was plugged with a butyl rubberstopper and the culture was freeze-dried using the same procedure asExample 1.

After freeze-drying, the seal on the vessel was broken and 3.0 ml ofsterile water was added to the vessel to rehydrate the culture. Uponproper incubation, the rehydrated culture exhibited substantiallogarithmic growth from an initial cell population of 9×10⁶ cells/ml.

The foregoing description of the present invention has focused primarilyon an embodiment wherein the freeze-dried preparation of microorganismsis rehydrated in the same vessel in which the preparation ofmicroorganisms was freeze-dried. In an alternative embodiment describedbelow, the freeze-dried preparation, containing at least concentratedmedium and microorganisms, is configured in a shape, e.g. a plug whichis removed from the container in which it is freeze-dried, stored inanother vessel and subsequently rehydrated. Rehydration is typicallyaccomplished by placing the plug in an amount or volume of watersufficient to bring the concentration of the medium to 1×. Accordingly,when the present invention is embodied in a plug, rehydration does nottake place in situ since the freeze-dried preparation is removed fromthe vessel in which it is freeze-dried and is rehydrated by placing theplug in a separate container with a volume of water sufficient to bringthe concentration of the medium to 1× which enables substantiallogarithmic growth of the viable microorganisms.

Providing freeze-dried preparations of microorganisms in plugs is knownin the art. The propagation of a culture using the prior art normally isaccomplished by inoculating freshly prepared growth media with all or apart of the rehydrated preparation. In other words, freeze-driedpreparations of microorganisms configured as plugs have been used topropagate fresh cultures using freshly prepared external nutrientmedium. In the present invention, the freeze-dried preparation in plugform is produced so that no external nutrient medium is required andwhen the plug is combined with an appropriate amount of watersubstantial logarithmic growth of the viable microorganisms will occur.

As explained above in the context of in situ rehydration, in the presentinvention, there is a relationship between the volume of the plug, themicroorganism concentration before freeze-drying, the volume of theculture desired upon rehydration and the concentration of the mediuminthe plug. For instance, if a 0.1 ml plug is formed, a 50× concentrationof medium would be needed to provide a rehydrated culture of 5.0 mlmedium of normal concentration. A 25× concentration of medium is neededto provide a rehydrated 5.0 ml culture if the plug has a volume of 0.2ml. If a 10.0 ml culture is desired from a 0.2 ml plug then 50×concentration of medium would be required. The relationship between theplug, microorganism concentration, medium concentration and volume ofthe culture desired upon rehydration is illustrated in Example 5.

Applicants have found that it is sometimes desirable to add a colloidalcompound to the cell suspension prior to freeze drying. The addition ofsuch colloidal compound provides a freeze dried preparation withenhanced mechanical characteristics suitable for formation of a plugwhich retains its shape after freeze-drying when the plug is removedfrom the container, e.g., the wells in which it was freeze-dried. Thephysical characteristics of some media, however, provide a plug withoutaddition of colloidal compounds. When addition of a colloidal compoundis desirable, however, the preferred colloidal compound for use withmost freeze-dried preparations is gelatin. The amount of colloidalcompound used should be selected to provide a plug which dissolves whenthe plug is placed in water. For gelatin-containing plugs, applicantshave found the use of from about 0.5% to 1.5% gelatin (w/v) of the cellsuspension prior to freeze-drying is preferred and use of about 1.0% ismost preferred. The techniques for using colloidal compounds such asgelatin to form a plug of freeze-dried microorganisms are known to thoseof ordinary skill in the art of freeze-drying microorganisms.

The following procedures may be followed to provide a freeze-driedpreparation in a plug configuration. To provide a cell suspension forfreeze-drying, a growth medium for the microorganism is provided inconcentrated form. The concentration of the medium is based upon theplug volume and the final volume of single strength medium desired uponrehydration. For example, if the plug volume is 0.2 ml then 25×concentration of medium is used for a 5.0 ml culture. The concentrationof the microorganism and the medium in the cell suspension is selectedto ensure that after freeze-drying substantial logarithmic growth willoccur upon rehydration with an amount of water sufficient to establishlogarithmic growth. The cell suspension is dispensed into the smallwells of a microtiter plate or other suitable device for forming a plugduring the freeze-drying process. Microtiter wells used in a laboratorycommonly hold 0.1 to 0.3 ml. The cell suspension is then freeze-dried ina conventional manner. After freeze drying the plugs are popped out ofthe microtiter plate aseptically and placed in vials or other suitablecontainers for storage. Several plugs may be contained within one vial.

To rehydrate a plug of the present invention, the plug is asepticallyremoved from its storage container and combined with a volume of steriledistilled water such that the resulting medium will be at singlestrength. Upon rehydration in an amount of water sufficient to provide amedium at single strength, substantial logarithmic growth of the viablemicroorganisms in the plug of freeze-dried microorganism will occur.

The plug configuration of applicants' invention is illustrated byExample 5 below. It must be understood, however, that the size of plugand the end volume of rehydrated material may be either smaller or muchlarger than the size and volumes used in Example 5 to illustrate theinvention. The upper limit on the size of plug is presently determinedprimarily by the practical and economic considerations associated withfreeze-drying large volumes of material and the extent to which mediumcan be concentrated.

EXAMPLE 5

The following test of the plug configuration was conducted with thebacterial strain Escherichia coli ATCC® 11775. Microtiter plates whichhad wells which would hold at least 0.2 ml were used. A final culturevolume of 5.0 ml was desired, thus a 25× concentration of nutrient brothmedium was used in the preparation. In this example, w designates theweight of the ingredient and v designates the volume of water presentprior to freeze-drying. The menstrum also contained 12% (w/v) sucrose,5% (w/v) bovine serum albumin fraction five and gelatin. Twoconcentrations of gelatin were tested 0.5% (w/v) and 1.0% (w/v). Theinitial microorganism concentration of 3.0×10⁹ cells/ml for the 0.5%gelatin preparation and 1.5×10¹⁰ cells of the 1.0% gelatin preparationrespectively. Upon rehydration for the 0.5% gelatin preparation therewere approximately 4×10⁷ viable cells per ml. For the 1.0% gelatinpreparation, there were approximately 2.0×10⁸ viable cells per ml. Afterfreeze-drying, plugs were removed from the microtiter wells. The plugswere then rehydrated by placing them in a container holding 5.0 mlsterile distilled water. After rehydration and 48 hours of growth themicroorganism concentration was 1.0×10¹² cells/ml for the 0.5% gelatinpreparation and 1.0×10¹¹ cells/ml for the 1.0% gelatin preparationrespectively.

While specific embodiments of this invention have been described herein,those skilled in the art will appreciate that changes and modificationsmay be made without departing from the spirit and scope of thisinvention, as defined in and limited only by the scope of the appendedclaims.

We claim:
 1. A freeze-dried preparation of microorganisms comprising apopulation of viable microorganisms which survive freeze-drying andnutrient medium in a sealed vessel, said nutrient medium added prior tofreeze-drying, wherein the population of viable microorganisms whichsurvive freeze-drying is determined prior to freeze-drying to ensurethat the population of viable microorganisms which survive freeze-dryingrelative to the concentration and volume of nutrients in said nutrientmedium is sufficient to support substantial logarithmic growth of thepopulation of viable microorganisms in said preparation upon rehydrationwith a fixed volume of water, wherein said fixed volume of water issufficient to initiate substantial logarithmic growth of said populationof viable microorganisms in said preparation and a sufficient amount ofcolloidal component to form said freeze-dried preparation ofmicroorganisms into a plug.
 2. A freeze-dried preparation as recited inclaim 1 wherein said colloidal compound is gelatin.
 3. A freeze-driedpreparation as recited in claim 2 wherein said gelatin is present insaid freeze-dried preparation in an amount from about 0.5% to 1.5% (w/v)of a cell suspension used to form said freeze-dried preaparation.
 4. Amethod of preparing a freeze-dried preparation of microorganismscomprising the steps of:a. growing a culture of the microorganisms in anutrient medium; b. forming a cell concentrate from said culture; c.determining the population of microorganisms which must survive freezedrying to provide a population of viable microoganisms afterfreeze-drying which will enter substantial logarithmic growth uponrehydration with a fixed volume of water, wherein said fixed volume ofwater is detemrined to be sufficient to initiate substantial logarithmicgrowth of said polulation of viable microorganisms; d. forming a cellsuspension of microorganisms for freeze-drying by adding freshconcentrated nutrient medium to said cell concentrsate whereby theconcentration and volume of nutrients in said nutrient medium relativeto the number of said microorganisms in said cell suspension provides acell population of viabIe microorganism after freeze-drying which willenter substantial logarithmic growth when said preparation is rehydratedin said vessel with said field volume of water; e. adding a suffcientamount of colloidal compound to said cell suspension to form a plug; andf. freeze-drying said cell suspension in a vessel to produce afreeze-dried preparation in said vessel.
 5. A method as recited in claim4 wherein said collodial compound is gelatin.
 6. A method as recited inclaim wherein said gelatin is present in said cell suspension in anamount from about 0.5% to 1.5% by weight of said cell suspension.